Identification of Flavonoids in Iraqi Date Palm Pollen by HPLC

Date palm pollen-DPP (Phoenix dactylifera L) plays an important role in traditional treatment in Iraq, especially for the treatment of fertility or as supplements, flavonoids were important components for date palm pollen, no information is available in the literature about the types of flavonoids in an Iraqi DPP variety El-Ghannmi Ahmar. The HPLC analysis revealed that in an Iraqi DPP variety El-Ghannmi Ahmar contains many types of flavonoids (13.590 μg/g lincoceric acid, 122.251 μg/ gIsorhamnetin,71.146 μg/g chlorogenic acid, 99.188 μg/g ferulic acid, 64.574 μg/g naringin, 109.117 μg/g apigenin, 48.391 μg/g apigenin-7-O-beta glycopyranoside, 28.883 μg/g letulin and 18.291 μg/g letulin-7-O-beta glycosides).


INTRODUCTION
Date Palm Pollen (Phoenix dactylifera, L.)-DPP is a fine, powder-like material produced by flowering plants and gathered by bees 1 , it is very important causing of human respiratory allergic disorders, involving the production of immunoglobulins and hence the release of histamine and other chemicals 2,3 .The early Egyptians and ancient Chinese used pollen as a rejuvenating medicinal agent.It has been called a "fountain of youth" 4 .Regular consumption of DPP is beneficial in nephropathy, rheumatism,gastropathy, sexual debilityand DPP extract have potential protective effects on testicular dysfunction induced by altered thyroid hormones 5,6 .In addition DPP grains have antioxidant and hypolipidemic effect in which exhibited significant reduction for serum cholesterol and low density lipoptoein-cholesterol(LDL-C), triglycerides and improve the level of high density lipoprotein-cholesterol( HDL-C), also the isolated flavonoids from DPP have anti atherosclerotic effects in high dose 7 .
Phytochemical screening showed that dried DPP contain sterols, flavonoids, triterpenoidal, saponins, tannins, and crude gonadotrophic substance [Egyptian cultivar] 2,8,9 , while in El-Ghannmi Ahmar DPP[Iraqi cultivar] phytosterols, alkaloids, protein, carbohydrates, glycosides, phenolic compounds, tannins, terpenoids, saponins, coumarins, lignin and flavonoids 10 .Many types of flavonoids has been identified in DPP, Abbas & Ateya 11 indicated five flavonoids compounds [rutin, luteolin -7-Ob -D -glucoside, apigenin, isorhamnetin-3-O-glucoside and naringin were isolated for the first time from the pollen.While Daoud et al found that the Tunisian cultivar contain higher concentrations of flavonoids than Kerkennah cultivar, which was about twice as high, especially in the acetone extract,and four types of flavonoids were identified in Tunisian cultivars by HPLC,which include Quercitin, Rutin, Catechin and Epicatechin 12 .So that the aim of this study is to identify the flavonoids inEl-Ghannmi Ahmar DPP[Iraqi cultivar].

Plant material
DPP (Phoenix dactylifera L.) variety El-Ghannmi Ahmar was collected from Samarra city,Salah Al-Din,Iraq separated from the kernels by fine gauze sieve and left in an incubator at 35°C for 3 hours.

Methods
Extraction and isolation of flavonoids (Ex-F) was done according to 13 method with some modification, while the chromatographic analyses for Ex-F was performed by HPLC (Shimadzu,10AV-LC,Japan) according to the method of the 14 with some modification by using C-18 column (150-4.6mm,5mm).The mobile phase consisted solvent of 0.1% acetic acid in deionized water and acetonitrile at a ratio (20:80).The UV detection wavelength and the flow rate were 264nm and 0.9 ml/min, respectively.In which isolated flavonoids was dissolved in methanol(HPLC grade) at a final concentration of 100 mg /ml and then filtered through a membrane filter (0.45 mm pore size) prior to injection.Twenty microliter of Ex-F sample was injected on C18-HPLC column.
Results are consistent with the previous study of 11 , which indicates the presence of many types of flavonoids in Egyptian DPP naringen, luteolin -7-Ob -D -glucoside, apigenin, isorhamnetin -3 -O-glucoside and rutin which isolated them by silica gel column chromatography and eluted by ethyl acetate, while the identification of these compounds were carried out by GC-MS and HPLC.Similarly, other study identified quercetin and rutin in DPP 8 .
No information were available in the literature about the DPP content of Isorhamnetin, luteolin, chlorogenic acid, ferulic acid and apigenin-7-O-beta glycopyranoside.The content of flavonoids in pollen may be affected by many enviermintal factors such as, air pollution, in which Rezanejad study the effect of air pollution on flavonoids in pollen grains of some ornamental plants, and found that HPLC analysis demonstrated that air pollution induces flavonoids accumulation to significantly higher levels in the polluted pollen of ornamental plants than in the control 15 .